Croton é um dos maiores gêneros de Euphorbiaceae, amplamente distribuído nas regiões tropicais do mundo. No Brasil existem cerca de 300 espécies, muitas utilizadas como medicinais no tratamento de diversos males, tais como úlceras, inflamações, malária, diabetes, sífilis e câncer. A variedade de usos medicinais reflete a diversidade química inerente ao gênero, cuja taxonomia também é bastante complexa. O objetivo deste trabalho é a caracterização química de sete espécies nativas de Croton, a fim de contribuir para a ampliação do conhecimento de potenciais farmacológicos e quiçá fornecer ferramentas quimiotaxonômicas que auxiliem na compreensão das relações entre grupos infragenéricos. Foram analisados óleos voláteis, flavonóides e componentes de extratos etanólicos brutos das espécies. Óleos voláteis de folhas de C. betulaster e C. glutinosus, e folhas e caules de C. hemiargyreus, C. antisyphiliticus, C. grandivelum, C. cf pycnocephalus e C. cf montevidensis foram extraídos por hidrodestilação em Clevenger e analisados por CG/EM. Todas as amostras apresentaram componentes voláteis, exceto uma amostra (folhas e caule) de C. antisyphiliticus, a espécie com folhas e caules menos aromáticos, e caules de C. grandivelum, de uma amostra de C. cf pycnocephalus e uma de C. hemiargyreus. Monoterpenos e sesquiterpenos são os componentes mais abundantes e não foram detectados fenilpropanóides. Caules apresentam em geral menos componentes voláteis que folhas. Os sesquiterpenos β-cariofileno, germacreno D e espatulenol são os mais comuns, enquanto 8-isoproprenil-1,5-dimetil-ciclodeca-1,5-dieno (C. betulaster e C. hemiargyreus), elixeno (em todas as espécies exceto C. betulaster e C. glutinosus) e o sesquiterpeno ledol (abundante em C. cf pycnocephalus) nunca foram antes relatados para Cróton. Extratos hidroalcoólicos das amostras foram analisados por CLAE e quando possível CLAE/EM e CLAE/EM/EM para detecção de flavonóides. Apenas em C. betulaster e C. hemiargyreus houve predominância de flavonas, tais como isoorientina, orientina, vitexina e diglicosídeos de apigenina. Nas demais espécies são abundantes glicosídeos de quercetina, como rutina e quercitrina, assim como tilirosídeo (glicosídeo acilado de campferol), que só não foi detectado em folhas de C. betulaster, C. glutinosus e C. cf montevidensis. A análise de outras espécies das mesmas seções (Barhamia e Codonocalyx, respectivamente) pode confirmar a coerência da ausência do tilirosídeo nas folhas como característica. Flavonóides metoxilados, como a casticina (em amostras de C. cf pycnocephalus, C. betulaster e C. glutinosus), também foram encontrados. Os extratos etanólicos brutos de todas as espécies exceto C. glutinosus foram analisados por CG/EM. Triterpenos, esteróides, alcalóides e ésteres de ácidos graxos são os componentes mais abundantes. Diterpenos não foram detectados. Benzoato de benzila é o componente majoritário dos extratos etanólicos brutos de C. betulaster, no qual também foram detectados cinamato de benzila, lupeol e lupenona Esteróides como sitosterol, campesterol e estigmasterol ocorrem em diversas espécies. A casticina é o componente majoritário dos extratos obtidos de folhas de C. cf pycnocephalus. Muitos alcalóides foram detectados em C. grandivelum, principalmente N-metil-crotonosina. Somente glaucina foi detectada em C. hemiargyreus. Os diferentes componentes dos extratos etanólicos das espécies estudadas possuem diversas atividades biológicas. As variações encontradas entre amostras de diferentes populações das mesmas espécies podem ser influenciadas por fatores ambientais ou genéticos. Com base nos dados obtidos, o isolamento e caracterização de flavonóides por EM e RNM, ensaios farmacológicos de acordo com as propriedades de cada grupo de substâncias e a caracterização de demais espécies das mesmas seções podem ser um interessante enfoque para estudos futuros e contribuir ainda mais para a compreensão da química e da taxonomia de Croton. / Croton is one of the largest genera of Euphorbiaceae, widely distributed in tropical regions of the world. There are around 300 species in Brazil, many used in traditional medicine to treat gastric ulcers, inflammations, malaria, diabetes, syphilis and cancer. The variety of medicinal applications reflects the diversity of biologically active compounds and the complexity of taxonomic relationships within the genus. The present work focuses on the chemical screening of seven Brazilian species of Croton, contributing to increase the knowledge about pharmacological potentials and hopefully provide chemotaxonomic tools to help understanding ralationships at the infrageneric level. Essential oils, flavonoids and compounds of crude ethanolic extracts were analysed. Esssential oils of leaves of C. betulaster and C. glutinosus, leaves and stems of C. hemiargyreus C. antisyphiliticus, C. grandivelum, C. cf pycnocephalus and C. cf montevidensis were extracted by hydrodistillation in Clevenger and analysed by GC/MS. Volatile compounds were found in all samples, except one sample (leaves and stems) of C. antisyphiliticus (the less aromatic among the species), and in stems of C. grandivelum, C. cf pycnocephalus and C. hemiargyreus. Monoterpenes e sesquiterpenes are major compounds and no phenylpropanoids were found. In general, leaves are richer in volatile compounds than stems. Sesquiterpenes such as β- caryophyllene, germacrene D and spathulenol are common, while 8-isoproprenyl-1,5- dimethyl-cyclodeca-1,5-diene (C. betulaster and C. hemiargyreus), elixene (in all species except C. betulaster and C. glutinosus) and the oxigenated sesquiterpene ledol (in C. cf pycnocephalus) are reported for Cróton for the first time. Hydroalcoholic extracts were analysed by HPLC and whenever possible also by HPLC/MS and HPLC/MS/MS to detect flavonoids. Flavones, such as isoorientin, orientin, vitexin and apigenin diglycosides were predominant only C. betulaster and C. hemiargyreus. Other species showed a profile richer in flavonols, such as quercetin glycosides (rutin, quercitrin and others) and tiliroside (kaempferol acylglycoside), which was not detected only in leaves of C. betulaster, C. glutinosus and C. cf montevidensis. Analyses of other species from the same sections (Barhamia e Codonocalyx, respectively) might confirm the coherence of the absence of tiliroside in leaves as a taxonomic character. Methoxylated flavonoids, such as casticin (found in samples of C. cf pycnocephalus, C. betulaster and C. glutinosus), were found. Crude ethanolic extracts of all species except C. glutinosus were analysed by GC/MS. Triterpenes, steroids, alkaloids and fatty acid esters were the predominant compounds of those extracts. Diterpenes were not detected. Benzyl benzoate predominates in crude ethanolic extracts from leaves of C. betulaster, along with benzyl cinnamate, lupeol e lupenone. Steroids such as sitosterol, campesterol and stigmasterol were found in several species. The flavonoid casticin is the major compound in extracts obtainded from leaves of C. cf pycnocephalus. Several alkaloids were detected in C. grandivelum extracts, mainly N-methyl-crotonosine. Glaucine was the only alkaloid identified in C. hemiargyreus. Several biological activities are known for the different compounds found in the crude ethanolic extracts. The differences between populations of the same species may be accounted for environmental or genetic influences. The data obtained suggest isolation and total structural determination of flavonoids by MS and NMR, pharmacological assays according to the properties of each group of compounds and chemical screening of remaining species of the same sections as interesting focuses for future studies, with the expectation to enhance the contribution to the chemistry and pharmacology of Croton and provide chemical synapomorphies for infrageneric groups within the genus.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Ternatina (5,4â-dihidroxi-3, 7, 8, 3â-tetrametoxi-flavona), um bioflavonÃide isolado de Egletes viscosa, Less (Compositae), foi estudado frente a vÃrios efeitos farmacolÃgicos em animais experimentais. O composto (15 e 30 mg/kg, i.p.) demonstrou de forma dose-dependente um efeito antiinflamatÃrio significante no edema de pata induzido por carragenina. Neste modelo, a indometacina (5 mg/kg, v.o.), uma conhecida droga antiinflamatÃria nÃo esterÃide, demonstrou maior potÃncia que a Ternatina. Com doses semelhantes Ãs anteriores, o bioflavonÃide reduziu efetivamente o aumento da permeabilidade capilar provocada por Ãcido acÃtico em camundongos, propriedade comum para muitos flavonÃides. Nesses mesmos animais, a Ternatina (15 e 30 mg/kg, i.p.) nÃo demonstrou atividade sobre o tempo de reaÃÃo no modelo da placa quente de Eddy (55Â1ÂC). PorÃm, no teste de âWRITHINGâ induzido por Ãcido acÃtico, o composto reduziu significativamente o nÃmero de contorÃÃes abdominais de maneira dose-dependente, indicando ter uma possÃvel propriedade analgÃsica perifÃrica. A substÃncia em estudo tambÃm mostrou atividade antipirÃtica em ratos no modelo de pirexia provocada por leveduras de cerveja. Este efeito, com dose de 30 mg/kg, via intraperitoneal, foi quase equivalente ao produzido por paracetamol (250 mg/kg, v.o.). A propriedade gastroprotetora da Ternatina foi evidenciada no modelo experimental de hipertermia gÃstrica aguda provocada por etanol 99% em camundongos. A Ternatina, nas doses de 30 e 60 mg/kg, via intraperitoneal, reduziu significativamente o desenvolvimento das mudanÃas hiperÃmicas induzidas por etanol no estÃmago. Essa droga (30 mg/kg, i.p.) tambÃm demonstrou um possÃvel efeito hepatoprotetor ao diminuir de forma significativa a atividade da alanina-aminotransferase (ALT) sÃrica nas lesÃes do fÃgado provocadas por tetracloreto de carbono (CCl4) em ratos. Nos animais tratados com Ternatina (30 mg/kg, i.p.) durante 8 dias consecutivos, o tempo de sono induzido por pentobarbital sÃdico foi significativamente maior que o do grupo controle, mostrando um provÃvel efeito depressor do flavonÃide sobre o metabolismo enzimÃtico de drogas no fÃgado e/ou em outros sistemas orgÃnicos. A Ternatina (15 e 30 mg/kg, i.p.) inibiu de forma dose-dependente o trÃnsito gastrointestinal em camundongos. AlÃm disso, o composto (10 e 40 Âg/mL no banho) produziu uma eficaz e reversÃvel inibiÃÃo das respostas contrÃteis causadas por diversos agonistas (acetilcolina, histamina, serotonina e cloreto de bÃrio) em Ãleo isolado de cobaio. A inibiÃÃo mostrou-se inespecÃfica e foi revertida por um aumento da concentraÃÃo de cÃlcio no banho, implicando que a Ternatina afeta os processos celulares dependentes desse Ãon. Em cÃlulas KBr, a substÃncia estudada evidenciou baixa citotoxicidade. A concentraÃÃo efetiva da droga necessÃria para inibir o crescimento celular (CyED50) foi de 100 Âg/mL. A Ternatina e o extrato bruto de Egletes viscosa, tambÃm exibiram baixa toxicidade geral. Os resultados obtidos no presente estudo como os compostos, juntamente com a atividade antiviral descrita contra poliovÃrus e adenovÃrus, sugerem que ela deve ser um dos principais componentes ativos de Egletes viscosa e, portanto, a base cientÃfica para comprovar o intensivo uso do extrato bruto da planta em medicina popular no tratamento dos distÃrbios gastrointestinais / Ternatin (5, 4â-dihydroxy-3, 7, 8, 3â-tetrametoxy-flavone), a flavonoid which was extracted and purified from Egletes viscosa, Less. (Compositae), was screened for various pharmacological effects in experimental animals. The compound (15 and 30 mg/kg, i.p.) demonstrated a dose-related significant anti- inflammatory effect in the carrageenan-induced rat hind paw test. In this model, indomethacin (5 mg/kg, p.o.), a known nonsteroidal anti-inflammatory drug (NASID), evidenced a much greater potency than to Ternatin. At similar doses, Ternatin effectively reduced the increase in vascular permeability induced by acetic acid in mice, a property common to many of the flavonoids. In mice, Ternatin (15 and 30 mg/kg, i.p.) failed to modify the reaction time in Eddyâs hot plate (55Â1ÂC) test, but in writhing test induced by acetic acid, it significantly reduced the number of abdominal contractions (writhes) in a dose-dependent manner indicating a possible peripheral analgesic property. Ternatin also showed antipyretic activity in the rat model of pyrexia induced by Brewerâs Yeast. Its activity at 30 mg/kg, i.p., was almost equivalent to that produced by paracetamol (250 mg/kg, p.o.). The gastroprotective effect of Ternatin was evident in the model of acute gastric hyperemia provoked by ethanol 99% in mice. Ternatin at doses of 30 and 60 mg/kg, i.p., significantly reduced the development of ethanol induced hyperemic changes in the stomach. Besides, Ternatin (30 mg/kg, i.p.) demonstrated hepatoprotective property by causing significant inhibition of carbon tetrachloride (CCl4) â induced increases in serum alanina-aminotransferase (ALT) in rats. In rats treated with Ternatin (30 mg/kg, i.p.) for eight consecutive days, the pentobarbital-induced sleeping times was found to be significantly higher than vehicle treated controls what means signifies the possible depressant effect of the compound on drug metabolizable enzymes in liver and/or other organ systems. Ternatin (15 and 30 mg/kg, i.p.) inhibited, in a dose-dependent manner the gastrointestinal propulsion in mice. In addition, Ternatin (10 to 40 Âg/mL in bath fluid) produced an effective reversible inhibition of contractile responses evoked by various agonists (acetylcholine, histamine, serotonin and barium chloride) in isolated guinea pig ileum. The inhibition was observed to be inespecific and could be overcome by an increase in the Ca++ concentration of bathing fluid implying that Ternatin affects cellular calcium dependent processes. In KB-cell lines, Ternatin evidenced low cytotoxicity. The effective drug concentration required to inhibit the growth of KB-cells (CyED50) was found to be 100 Âg/mL. Ternatin and the crude extrats of Egletes viscosa evidenced low order of general toxicity. The present findings on Ternatin, together with the reported antiviral activity against polioviruses and adenoviruses suggest that it might be the unique active compound present in Egletes viscose and this may be the scientific basis for the extensive use of plant crude extracts in popular medicine for treating gastrointestinal disturbances

No presente trabalho estudou-se por métodos analíticos (CCD e CLAE/UV) a atividade antioxidante dos flavonóides presentes na cana-de-açúcar (Saccharum officinarum L.), visando sua possível utilização como alimento funcional. As técnicas CLAE/UV e microfracionamento por CLAE permitiram o fracionamento e o isolamento dos flavonóides, os quais foram analisados por CCD utilizando reagentes específicos para detecção de substâncias antioxidantes. Os resultados obtidos permitiram identificar os flavonóides da cana-de-açúcar (folhas e garapa) com atividade antioxidante, através da comparação dos espectros de UV/DAD e tempo de retenção: orientina-O-ramnosídeo nas folhas e schaftosídeo, isoschaftosídeo, diosmina-8-C-glicosídeo, orientina e 4\',5\'-di-O-metil-luteolina-8-C-glicosídeo na garapa. Estes resultados justificam estudos nutricionais e/ou farmacológicos mais aprofundados a fim de classificar (ou não) a cana-de-açúcar como alimento funcional. / In the present study, analytical methods (TLC and HPLC/UV) were used to evaluate the antioxidant activity of the flavonoids of sugar cane (Saccharum officinarum L.) regarding to its possible use as a functional food. HPLC/UV and HPLC microfractionation techniques allowed fractionation and isolation of the flavonoids, which were evaluated by TLC using specific reagents to detect the antioxidant compounds. The results allowed to identify the flavonoids of sugar cane with antioxidant activity, based on comparison of UV-DAD spectra and retention time: orientin-O-rhamnoside in the leaves and schaftoside, isoschaftoside, diosmetin-8-C-glycoside, orientin and 4\'-5\'-dimethyl-luteolin-8-C-glycoside in the juice. Theses results justify more detailed nutricional and pharmacologic studies to classify (or not) sugar cane as a functional food.

\"Maracujá\" é o nome popular de várias espécies, dentre as quais Passiflora edulis e Passiflora alata (Passifloraceae). Esta última é uma planta medicinal amplamente utilizada no Brasil devido às suas propriedades ansiolíticas e sedativas, sendo também a espécie oficial da Farmacopéia Brasileira. Porém, freqüentemente ocorre a sua substituição indevida por Passiflora edulis, espécie usada para o preparo de sucos. Há também a confusão com a Passiflora incarnata, espécie usada na Europa, mas que não se adapta bem ao clima brasileiro. Neste trabalho foi desenvolvido um método espectrofotométrico por UV-visível para a quantificação dos flavonóides totais contidos nas folhas de Passiflora alata e de Passiflora edulis, a partir da adaptação das metodologias descritas nas Farmacopéias Francesa, Helvética e Européia para a espécie Passiflora incarnata. O procedimento proposto envolveu a complexação dos flavonóides com cloreto de alumínio e foi utilizada a rutina como padrão. Confrontou-se os dados obtidos pelo método espectrofotométrico desenvolvido neste trabalho com os obtidos por HPLC. Os melhores resultados foram obtidos com a metodologia adaptada da Farmacopéia Francesa. / \"Maracujá\" is the popular name of some species, among which are Passiflora edulis and Passiflora alata (Passifloraceae). The latter is a widely used medicinal plant in Brazil due to its ansiolytic and sedative properties, and also the official species of the Brazilian Pharmacopoeia. However, it is quite often improperly substituted for Passiflora edulis, which is used for the preparing juices. Another inadequate substitution occurs with Passiflora incarnata, wich is used in Europe, but does not adjust to the Brazilian climate. From adapting the methodologies described in the French, Helvetian and European Pharmacopoeia for the Passiflora incarnate species, a spectrophotometric method by UV-Visible has been developed to enable quantification of all flavonoids comprised in the leaves of both Passiflora edulis and Passiflora alata. The suggested procedure involved the complexation of the flavonoids with aluminum chloride, being rutin used as standard. Data obtained by the spectrophotometric method developed in the present work were compared with those obtained by HPLC. The French Pharmacopoeia methodology provided the most significant data in the study.

Orientador: Gonçalo Amarante Guimarães Pereira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T10:55:22Z (GMT). No. of bitstreams: 1 Lepikson-Neto_Jorge_D.pdf: 134833000 bytes, checksum: 09f83f4cd8ca673cb66cad9748effe06 (MD5) Previous issue date: 2013 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital. / Abstract: The abstract is available with the full electronic document. / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Magister Pharmaceuticae - MPharm / Artemisia afra (A. afra) is traditionally used for a variety of ailments and contain flavonoids e.g. luteolin which may contribute to some of its activity. It is generally administered as a tea or decoction, and such liquid dosage forms present challenges as far as long term storage and stability are concerned, as well as sub-optimal oral bioavailability of actives they contain. Freeze dried aqueous extracts (FDAE) can alleviate such problems but may be hygroscopic and unstable. The use of modified forms of FDAE can counter the problem of hygroscopicity (e.g. use of alginate) and alleviate the issue of sub-optimal bioavailability of plant actives (e.g. polymethylmethacrylate). The objectives of this study, were to: (1) prepare the freeze dried aqueous extract (FDAE) and modified forms, which include alginate-extract beads (alginate-FDAE) and polymethylmethacrylate coated alginate matrix beads of herbal extract (PMMA-alginate-FDAE) of the FDAE of A. afra, (2) determine and compare the pharmaceutical characteristics of the above mentioned preparations of A. afra,(3) quantify and compare the total flavonoid and specifically luteolin levels of the different forms of A. afra,(4) evaluate and compare the release characteristics of FDAE of A. afra from the alginate-FDAE and PMMA-alginate-FDAE beads in gastrointestinal fluids and (5) determine the intestinal permeability of luteolin contained in selected modified Artemisia afra extract preparations. It was hypothesized that making the alginate beads and the polymethylmethacrylate coated alginate beads would make the FDAE less hygroscopic with a lower moisture content, that the rate of release of luteolin from A. afra FDAE into gastrointestinal fluids would be faster than from the modified forms, and that the effective gastrointestinal permeability of luteolin in the alginate-FDAE and PMMA-alginate-FDAE beads of A. afra is equal to that in FDAE. To realize these objectives, the FDAE was prepared by freeze drying the aqueous extract of the A. afra dried leaves, alginate-FDAE prepared by dispersing FDAE into 4% sodium alginate solution, then adding the resulting stock solution into a 2% calcium chloride solution and drying resulting beads and PMMA-alginate-FDAE prepared by a modified water-in-oil-in-water emulsion solvent evaporation method using water as an internal aqueous phase. Using pharmacopoeial methods and methods adapted from other workers the organoleptic and pharmaceutical characteristics were determined to compare the pharmaceutical quality of these preparations of A. afra. To identify and determine the levels of luteolin in the plant preparations, a validated HPLC assay was developed. Finally, the in situ perfused rat intestine model was used to determine the in vitro bioavailability, i.e. gastrointestinal permeability, of luteolin from solutions containing luteolin in pure form, FDAE, alginate-FDAE and PMMA-alginate-FDAE. The A. afra forms were obtained in moderate to good yields and FDAE was brown and hygroscopic in nature, the alginate beads dark brown free flowing and spherical in shape and the PMMA-alginate beads light brown in colour with rough edges. The A. afra plant forms on average contained 0.185 ± 0.24, 0.067 ± 0.014, 0.012 ± 0.071 μg/mg of free luteolin (n=3) in FDAE, alginate-FDAE and PMMA-alginate-FDAE respectively and 0.235 ± 0.026, 0.079 ± 0.093, 0.058 ± 0.082 μg/mg of total luteolin (n=3) in FDAE, alginate-FDAE and PMMAalginate- FDAE respectively. The Plumen values for intestinal uptake of luteolin were significantly higher from solutions of A. afra forms than the pure luteolin solution (i.e. Plumen values in the range of 0.02 - 0.035 cm/s for all plant forms vs Plumen values in the range of 0.010 - 0.014 cm/s for pure luteolin, t-test p = 0.0252). The permeability of luteolin in FDAE appeared to be slighter greater than that of the modified forms (Plumen values >0.03 cm/s for FDAE and Plumen values <0.03 cm/s for both modified forms). In summary, the results showed that, the modified A. afra forms; alginate-FDAE and PMMAalginate- FDAE were of acceptable pharmaceutical quality with luteolin better taken up in the plant forms than in its pure form. The A. afra forms prepared had similar rates of uptake (permeability) of free and total luteolin with the rates being highest for the FDAE. Collectively, these results indicate that alginate-FDAE and PMMA-alginate-FDAE bead forms should be suitable for use in a solid dosage form (e.g. tablet or capsule) of A. afra.

Orientador: Filipe Pereira Giardini Bonfim / Banca: Nathalia de Souza Parreiras / Banca: Jordany de Oliveira Gomes / Resumo: O manejo cultural de plantas medicinais é fator que influencia fortemente na síntese de metabólitos secundários, sendo a adubação fundamental para alcançar boa produtividade e teores satisfatórios de compostos bioativos. Dessa forma, o objetivo do estudo foi avaliar a produção de capítulos florais, compostos fenólicos totais, flavonoides totais, bem como o crescimento de calêndula (Calendula officinalis L.) submetida a tempos de incorporação de torta de mamona em diferentes épocas de cultivo. O trabalho foi divido em dois capítulos, no primeiro avaliou-se a massa fresca e seca dos capítulos florais, número de capítulos florais, teor de compostos fenólicos e de flavonoides de capítulos florais em três tempos de incorporação de torta mamona (28 e 14 dias antes do transplantio e no dia do transplantio) associados a quatro épocas de cultivo. A análise dos dados revelou que a produção de material vegetal foi influenciada tanto pelo tempo de incorporação da torta de mamona quanto pela época de cultivo, apresentando interação entre os fatores. Na época de cultivo 1 médias inferiores foram verificadas com a incorporação 0 DAT. Menores médias foram encontradas também com incorporação 28 DAT na época de cultivo 3. Nas épocas de cultivo 2 e 4 não houve diferença estatística entre os tempos de incorporação, entretanto a época de cultivo 2 apresentou valores inferiores de produção de material vegetal quando comparada às demais épocas. Deste modo, não é indicado o cultivo em condições clim... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The cultural management of medicinal plants is a factor that strongly influences the synthesis of secondary metabolites, with the fertilization being fundamental to achieve good productivity and satisfactory levels of bioactive compounds. Thus, the objective of the study was to evaluate the production of floral chapters, total phenolic compounds, total flavonoids, as well as the growth of calendula (Calendula officinalis L.) submitted to times of incorporation of castor bean cake at different growing seasons. The work was divided in two chapters, in the first one was evaluated the fresh and dry mass of the floral chapters, number of floral chapters, content of phenolic compounds and flavonoids of floral chapters in three times of incorporation of castor bean cake (28 and 14 days before transplanting and on the day of transplanting) associated with four growing seasons. The data analysis has revealed that the production of plant material was influenced both by the incorporation timeof the castor bean cake and the growing season, showing interaction between the factors. At the time of cultivation 1 lower means were verified with the 0 DAT incorporation. Minor means were also found with 28 DAT incorporation at the time of cultivation 3. In the growing seasons 2 and 4 there was no statistical difference between the incorporation times, however the crop season 2 presented lower values of plant material compared to the other. Thus, cultivation is not indicated in climatic conditions similar to those of the growing season 2 (high rainfall and high temperatures) due to the low productivity of inflorescences. The amount of phenolic compounds did not present significant differences depending on the treatments. Lower mean values in the flavonoid amount were observed with the incorporation of 28 days before the transplanting in the growing season 4. In Chapter 2, the influence of three incorporation ... / Mestre

Orientador: Marinônio Lopes Cornélio / Banca: Hamilton Cabral / Banca: Marcelo Andrés Fossey / Resumo: Os flavonóides são uma grande classe de polifenóis ocorrendo de forma natural amplamente distribuídos nas plantas, essas moléculas exibem algumas atividades farmacológicas importantes como anti-inflamatória, antioxidante, anticancerígena e antibacteriana. A interação entre os flavonóides Rutina (Ru) e Guaijaverina (Gua) e a Albumina do Soro Humano (HSA) em pH 7,0 fisiológico foi investigado usando a técnica de espectroscopia de fluorescência de estado estacionário, cálculo ab initio e de modelagem molecular. A partir da supressão de fluorescência da HSA pelos flavonóides, a constante de supressão de Stern-Volmer ( SVK ) e sua forma modificada ( aK ) foram calculadas em 298, 303 e 308 K, como também os parâmetros termodinâmicos correspondentes H, G e S, para cada flavonóide. A análise do equilíbrio de ligação foi utilizada para determinar os valores do número de sítios de ligação ( n ) e a constante de ligação ( bK ) para a Rutina e a Guaijaverina em 298, 303 e 308 K. A distância média entre o doador (HSA-214Trp ) e o aceitador (Ru e Gua) foi estimada de acordo com a teoria de transferência de energia ressonante fluorescente. A otimização geométrica dos flavonóides Rutina e Guaijaverina foi realizada nos seus estados fundamentais usando o funcional DFT/B3LYP ab initio com um conjunto de bases 6-31G(d,p) utilizado nos cálculos. O cálculo de modelagem molecular indica que os flavonóides se localizam no sítio I da HSA, dentro do bolso hidrofóbico do subdomínio IIA. Os resultados teóricos obtidos pela modelagem molecular corroboram com os dados de espectroscopia de fluorescência / Abstract: Flavonoids are a large class of naturally occurring polyphenols widely distributed in plants, these molecules exhibit some important pharmacological activities like anti-inflammatory, antioxidant, anticancer, and antibacterial. The interaction between Rutina and Guaijaverin flavonoids and Human Serum Albumin (HSA) at physiological pH 7.0 was investigated by using the technique of fluorescence spectroscopy, ab initio and molecular modeling calculation. From the fluorescence quenching of the HSA by flavonoids, the Stern-Volmer quenching constant ( SVK ) and its modified form ( aK ) were calculated at 298, 303 and K308, as well as the corresponding thermodynamic parameters H, G and S, for each flavonoid. Analysis of binding equilibria was utilized to determine the number of binding sites ( n ) and binding constants ( bK ) values for Rutin and Guaijaverin at 298, 303 and K308 . The average distance between donor (HSA-214Trp ) e acceptor (Ru and Gua) was estimated according to the theory of fluorescence resonance energy transfer. The geometry optimization of Rutin and Guaijaverin flavonoids was performed in its ground state by using ab initio DFT/B3LYP functional with a 6-31G(d,p) basis set applied in calculations. Molecular modeling calculation indicated that the flavonoids are located in site I of HSA, within the hydrophobic pocket of the subdomain IIA. The theoretical results obtained by molecular modeling are corroborated by the fluorescence spectroscopy data / Mestre

Flavonoids are a specialized group of compounds produced by plants that give them greater adaptability to their environment and ultimately enhance their ability to survive. In plants, one function of flavonoids is to attract pollinators by their various flavor and scent profiles. They also protect the photosynthetic machinery from photo-oxidation. In humans, flavonoids have been shown to act as antioxidants, exhibit antimicrobial activity, and have shown potential as cancer treatments. In nature, flavonoids are most often found coupled with a sugar group (glucose, rhamnose, and others) which imparts stability and increases bioactivity. The process of adding a sugar (glycosylation) is catalyzed by a class of enzymes called glycosyltransferases (GT). One such enzyme found in grapefruit only glucosylates the flavonol class of flavonoids at the 3-OH position and is of interest due to its unique substrate and regio-specificity. Called Cp3GT (Citrus paradisi flavonol 3-O glucosyltransferase), this enzyme is similar in structure to other plant GT’s yet differs in the flavonoids it can glucosylate and where the glucose can be added. To date, the literature has not reported a structural mechanism for a flavonol specific 3-O glucosyltransferase’s unique catalytic activity. High-resolution structural imagery of enzymes, elucidated using X-ray crystallography, can be used to direct custom enzyme development to produce bioavailable natural products. Furthermore, structural research on enzymes with high specificity strengthens enzyme-ligand docking simulations, which are commonly used to test the binding affinity of potential pharmaceuticals. This research hypothesizes Cp3GT has structural features that confer its unique substrate and regiospecificity that are not revealed by homology modeling. This hypothesis will be tested using x-ray crystallography of purified Cp3GT protein bound to its preferred flavonol substrates. The gene for Cp3GT was transformed into Pichia pastoris and was recombinantly expressed using methanol induction. Cp3GT was purified to 80% purity using cobalt metal affinity chromatography. Cp3GT was subjected to additional purification measures using anion exchange chromatography with the goal of increasing purity to ≥95% for crystallization experiments. Purity analysis was conducted using SDS-PAGE (Coomassie/silver stain, western blot) and UV-Vis spectrophotometry. While initial results are promising, additional purification steps may be needed to achieve the purity necessary for crystallization.

This investigation attempts to verify the identification of the anthocyanin in A. amplectens by spectral means. An attempt was also made to identify the sugars, and possibly their locations on the heterocyclic ring system, of the anthocyanins.